TRPV4 dysfunction in endothelial cells from STZ treated rats reversed by insulin.

Yousif Shamsaldeen, Lisa Lione, Christopher Benham

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Abstract

Diabetes mellitus is a pandemic metabolic disorder characterized by chronically elevated blood glucose concentration (hyperglycaemia) that contributes to vascular complications (1). Endothelial dysfunction is a major complication resulting in impaired vasodilation rendering the diabetic prone to elevated blood pressure (1). The vanilloid transient receptor potential channel TRPV4 is expressed in vascular endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) (2, 3). Endothelial TRPV4 is sensitive to shear stress and mediates endothelial calcium influx transduced to VSMC hyperpolarization and hence vasodilation (4, 5). In this study the effect of insulin was investigated on TRPV4 dysfunction in endothelial cells from STZ treated rats.
Two groups of male Wistar rats (N= 4-5/group) were studied, the first group was injected i.p. with 65mg/kg streptozotocin (STZ) while the second was injected with 20mM citrate buffer (Control). Thoracic aortic rings from rats that had been euthanized, were isolated and suspended in organ baths attached to a transducer allowing quantitative isometric tension measurement. Arterial rings were pre-contracted with noradrenaline (300nM) followed by TRPV4-agonist, 4αPDD (3pM-3µM) to evoke relaxation dose response curves. In addition, primary ECs were isolated from thoracic aorta. The cells were then studied through Fura-2 calcium imaging to measure the calcium influx induced by 4αPDD and TRPV4 protein levels were probed with rabbit Anti-TRPV4 antibody and visualized through confocal immunocytochemistry.
Thoracic aortic rings from diabetic rats showed significantly less TRPV4-induced maximum relaxation compared to control (*P˂0.05 Diabetic: 56.0±5.5% vs control; 81.1±2.1%). Endothelial cells treated with 4αPDD (30µM) showed significant reduction in TRPV4 mediated calcium influx (Diabetic: *P˂0.05; 49.0±12.6% vs control 100±15.9%) and significant delay in response (**P˂0.01; 247.1±23.5 s vs control 123.7±11.1 s). These findings were accompanied with significant decrease in diabetic primary endothelial cells TRPV4 expression which was reversed with insulin (270mIU/day for 5 days (Diabetic: **P˂0.01, 58.9±5.8% vs diabetic treated with insulin: 95.4±5.4% and ***P˂0.001 vs control 100±8.8%). Additionally, ECs showed significant reduction in caveolin-1 (Diabetic: **P˂0.01, 73.9±4.3% vs diabetic treated with insulin: 103.1±3.8% and *P˂0.05 vs control 100±7.2%).
These results showed the TRPV4 function is compromised in endothelial cells and may be in part due to reduction in channel number. Endothelial TRPV4 reduction was accompanied with caveolin-1 reduction. Caveolin-1 and eNOS reduction was previously reported in diabetic kidney and endothelial cells which was restored by insulin stimulated translocation of protein to the cell membrane (6, 7). Our results suggest that TRPV4 translocation may also be insulin sensitive. Therefore, insulin might provide direct vascular protection in diabetics in addition to regulating glucose metabolism.

1) Guariguata et al. (2014). Diabetes Research and Clinical Practice 103: 137-149.
2) Kwan H et al. (2007). Biochimica et Biophysica Acta, 1772: 907-914.
3) Watanabe H et al. (2008). Pharmacology and Therapeutics, 18: 337-351.
4) Earley S et al. (2004). Circ Res, 95: 922-929.
5) Freichel M et al. (2005). JPhysiol, 567.1: 59-66.
6) Komers R et al. (2006). Diabetes, 55: 1651-1659.
7) Wang H et al. (2009). Mol Endocrinol, 23: 1613-1623.


Original languageEnglish
Title of host publicationNew Therapeutics for Diabetes and Obesity Keynote symposium, California
Publication statusPublished - 17 Apr 2016

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